The goal of this proposal is to develop novel methods to generate chemical and biochemical tools suitable for probing the physiological effects of lysine post-translational modifications (PTMs). Protein PTMs play a significant role in epigenetic regulation and are known to occur at different rates in aberrant cells as opposed to normal cells, implicating them as major players in disease states such as cancer. While well-known PTMs such as acetylation and methylation have been extensively studied, little is known regarding more recently identified PTMs such as succinylation and hydroxybutyrylation. Therefore, there is a substantial need for new techniques that will allow for their efficient evaluation. While a few methods for introducing unnatural and modified amino acids exist, they are all severely limited by yield, versatility of site and chemical scope. The first two specific aims presented in this application are designed to address this issue. Aim 1. Develop cysteine alkylation strategies to incorporate site specific acyl-lysine mimics into protein targets. Cysteine alkylation offers a versatile way to chemically introduce these mimics into proteins and, if successful, our approach would be applicable to mimicking other PTMs. The generation of histones containing a succinylation mimic and phosphoglucose isomerase (PGI) containing a hydroxybutyrylation mimic will serve as representative examples of method feasibility. Aim 2. Evaluate the effect of specific lysine PTMs on cellular functions. Using the mimics prepared in Aim 1, the effect of histone lysine succinylation on nucleosome assembly and of PGI hydroxybutyrylation on protein activity will be determined with kinetic assays, Western blot analysis and ratiometric fluorescence assays. Changes in protein-protein interactions will also be evaluated using standard pull-down and Co-IP assays. Aim 3. In addition, histone lysine methylation will also be investigated using 15-20 rationally designed small molecule probes to inhibit lysine specific demethylase 1 (LSD1), one of the enzymes responsible for removing histone methylation marks. Based on preliminary findings, a series of mechanism based inactivators that covalently bind to the associated flavin cofactor have been proposed. Their effect on enzymatic activity can be evaluated using previously described kinetic assays and Western blot experiments in addition to cell growth and migration assays. The work outlined in this proposal promises to yield broadly applicable methods for evaluating protein PTMs and provide substantial insight into epigenetic regulation and its relation to diseases such as cancer. In addition, the proposed research training plan will provide the applicant with a unique opportunity to significantly expand his experimental repertoire to include biochemical and cell culture techniques. Johns Hopkins University provides a scientifically rich and collaborative atmosphere that should encourage the applicant's development. An array of supplementary educational and teaching opportunities should facilitate the applicant's launch toward a successful career as an independent investigator.